Though latently infecting most of us asymptomatically, human cytomegalovirus (HCMV) is a leading viral cause of birth defects and can be life-threatening to immune-compromised individuals. As a member of the ?- herpesvirus subfamily of the Herpesviridae and the most structurally and genetically complex herpesvirus (e.g., its genome is twice that of chickenpox-causing varicella zoster virus, an ?-herpesvirus), HCMV is one of the largest of all viruses and presents a major challenge to structure determination. Architecturally similar to other herpesviruses, HCMV is composed of a glycoprotein-containing envelope, a tegument layer, and a bacteriophage-like icosahedral capsid enclosing a genome of a single dsDNA molecule. Distinctive from members of the ?- and ?-herpesvirus subfamilies are two processes central to HCMV infection: 1) its large genome needs to be packaged through a portal complex and then stabilized by a unique tegument protein pp150; 2) the process of cell fusion by gB involves a unique pentameric glycoprotein complex gH/gL/UL128/UL130/UL131. These processes thus can be targeted for structure-guided design for novel vaccines and anti-virals against HCMV infections. By cryo electron microscopy (cryoEM), the PI?s group obtained the first three-dimensional structure of HCMV capsid at 18 resolution in 1999, which was followed by progressive improvement in resolution, culminating at the recent 3.9 resolution structure reported in Science. Our pilot studies resolved the portal complex and pp150-capsid interactions absent from ?- and ?-herpesvirus subfamilies. Furthermore, we have demonstrated atomic resolution structure determination for membrane protein complexes and?in collaboration with Merck?obtained preliminary cryoEM data for HCMV pentameric glycoprotein complexes. We hypothesize that our state-of-the-art technologies in electron-counting cryoEM, symmetry relaxation and local refinement methods, and HCMV BAC technologies together would now allow us to determine in situ structures of genome packaging/ejection portal complex, pp150 and glycoprotein complexes, and when combined with structure-guided mutagenesis, to identify essential hot-spot residues critical to the interactions among these proteins. Harnessing technology breakthroughs in cryoEM and structure-guided mutagenesis, the proposed research aims to: (1) obtain in situ structure of the portal of DNA genome packaging and ejection machinery at near-atomic resolution and identify residues critical to capsid assembly and stabilization; (2) determine the in situ structure of pp150 at about 2 resolution and identify the chemical bonds between capsid and capsid-interacting pp150 residues, particularly the cys tetrad conserved among primate cytomegaloviruses; (3) obtain atomic structures of purified pentameric glycoprotein complex in complex with three neutralizing monoclonal antibodies, as well as their in situ pre-fusion glycoprotein structures on viral envelope by cryo electron tomography for comparison. The expected results should inform efforts in designing inhibitors and vaccines against HCMV infections.